Yi-Quan Wang

Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes

Vertebrates diverged from other chordates ~500 Myr ago and experienced successful innovations and adaptations, but the genomic basis underlying vertebrate origins are not fully understood. Here we suggest, through comparison with multiple lancelet (amphioxus) genomes, that ancient vertebrates experienced high rates of protein evolution, genome rearrangement and domain shuffling and that these rates greatly slowed down after the divergence of jawed and jawless vertebrates. Compared with lancelets, modern vertebrates retain, at least relatively, less protein diversity, fewer nucleotide polymorphisms, domain combinations and conserved non-coding elements (CNE). Modern vertebrates also lost substantial transposable element (TE) diversity, whereas lancelets preserve high TE diversity that includes even the long-sought RAG transposon. Lancelets also exhibit rapid gene turnover, pervasive transcription, fastest exon shuffling in metazoans and substantial TE methylation not observed in other invertebrates. These new lancelet genome sequences provide new insights into the chordate ancestral state and the vertebrate evolution.

Branchiostoma japonicum and B-belcheri are distinct lancelets (cephalochordata) in xiamen waters in China

Abstract Lancelets in Xiamen were reported as Branchiostoma belcheri in 1932, and subsequently were believed to comprise a single species. However, recent studies revealed that Xiamen lancelets actually represent two species, B. belcheri and B. japonicum. We observed thousands of lancelets from Xiamen beach to recognize these two species. Our observations showed that at least three morphological characters distinguish them: 1) the rostral fin is slightly round with the end obtuse in B. belcheri but elliptic with the end cuspate in B. japonicum; 2) the number of preanal fin-chambers is more than 80 in B. belcheri but less than 64 in B. japonicum, and the chambers are slender in the former but stout in the latter; 3) the caudal fin of B. belcheri is narrower than that of B. japonicum, and the angle between the dorsal and super-caudal fins, and between preanal and sub-caudal fins, is obtuse in B. belcheri but acute in B. japonicum. We also provide some ecological and distributional evidence to support the conclusion that there are two separate species in Xiamen waters.

The Evolutionarily Dynamic IFN-Inducible GTPase Proteins Play Conserved Immune Functions in Vertebrates and Cephalochordates

Interferon (IFN)-inducible GTPases currently include four families of proteins: myxovirus resistant proteins (Mxs), guanylate-binding proteins (GBPs), immunity-related GTPase proteins (IRGs), and very large inducible GTPase proteins (VLIGs). They are all under conserved regulation by IFNs in humans and mice and play a critical role in preventing microbial infections. However, differences between vertebrates are poorly characterized, and their evolutionary origins have not been studied in detail. In this study, we performed comparative genomic analysis of the four families in 18 representative animals that yielded several unexpected results. Firstly, we found that Mx, GBP, and IRG protein families arose before the divergence of chordate subphyla, but VLIG emerged solely in vertebrates. Secondly, IRG, GBP, and VLIG families have experienced a high rate of gene gain and loss during the evolution, with the GBP family being lost entirely in two pufferfish and VLIG family lost in primates and carnivores. Thirdly, the regulation of these genes by IFNs is highly conserved throughout vertebrates although the VLIG protein sequences in fish have lost the first 870 amino acid residues. Finally, amphioxus IFN-inducible GTPase genes are all highly expressed in immune-related organs such as gill, liver, and intestine and are upregulated after challenge with PolyI:C and pathogens, although no IFNs or their receptors were detected in the current amphioxus genome database. These results suggest that IFN-inducible GTPase genes play conserved immune functions both in vertebrates and in cephalochordates.

Characterization of microRNAs in cephalochordates reveals a correlation between microRNA repertoire homology and morphological similarity in chordate evolution

SUMMARY Cephalochordates, urochordates, and vertebrates comprise the three extant groups of chordates. Although higher morphological and developmental similarity exists between cephalochordates and vertebrates, molecular phylogeny studies have instead suggested that the morphologically simplified urochordates are the closest relatives to vertebrates. MicroRNAs (miRNAs) are regarded as the major factors driving the increase of morphological complexity in early vertebrate evolution, and are extensively characterized in vertebrates and in a few species of urochordates. However, the comprehensive set of miRNAs in the basal chordates, namely the cephalochordates, remains undetermined. Through extensive sequencing of a small RNA library and genomic homology searches, we characterized 100 miRNAs from the cephalochordate amphioxus, Branchiostoma japonicum, and B. floridae. Analysis of the evolutionary history of the cephalochordate miRNAs showed that cephalochordates possess 54 miRNA families homologous to those of vertebrates, which is threefold higher than those shared between urochordates and vertebrates. The miRNA contents demonstrated a clear correlation between the extent of miRNA overlapping and morphological similarity among the three chordate groups, providing a strong evidence of miRNAs being the major genetic factors driving morphological complexity in early chordate evolution.

The effects of encapsulating C60 fullerenes on the bending flexibility of carbon nanotubes

We investigate the bending flexibility of carbon nanotubes (CNTs) with encapsulated C60 fullerenes, using molecular dynamics (MD) simulations. Our simulations on the bending of the fully ((C60)12@(10,10)) and partially ((C60)10@(10,10)) filled peapods show an 18 and 6.3% increase of the flexural rigidity, and a 45 and 11% increase of the buckling strength, respectively, compared to the empty (10, 10) CNT. What is characteristically different for the peapod from the empty CNT is the presence of a transitional region in the loading process that proceeds to the onset of buckling. Within this transitional region, the interaction between the encapsulated fullerenes and the hosting CNT leads to an unusual configuration of the peapod, in which there are ripples along the inner arc of the bent peapod. The transition region in the partially filled peapod is short compared with the fully filled peapod. This is mainly caused by the axial motion of C60 fullerenes, especially after the appearance of the small ripple. The rippling configuration has been reported previously in the bending of multi-walled CNTs, where it emerges after the critical bending angle. However, in the present case, the peapod remains perfectly elastic in this transitional region until buckling takes place.

Consecutive Spawnings of Chinese Amphioxus, Branchiostoma belcheri, in Captivity

Cephalochordate amphioxus is a promising model animal for studying the evolutionary and developmental mechanisms of vertebrates because its unique phylogenetic position, simple body plan and sequenced genome. However, one major drawback for using amphioxus as a model organism is the restricted supply of living embryos since they are available only during spawning season that varies from a couple of days to several months according to species. Therefore we are aiming to develop methods for obtaining viable amphioxus embryos in non-spawning season. In the current study, we found that Branchiostoma belcheri could develop their gonads and spawn consecutively in the laboratory when cultured in a low density at a high temperature (25–28°C) supplied with sufficient food and proper cleanness. Among the approximate 150 observed animals, which spawned spontaneously between November and December 2011, 10% have spawned twice, 10% three times, and 80% four times, through April 2012. The quality and quantity of the gametes reproduced in the consecutive spawning have no obvious difference with those spawned once naturally. Spawning intervals varied dramatically both among different animals (from 1 to 5 months) and between intervals of a single individual (from 27 to 74 days for one animal). In summary, we developed a method with which, for the first time, consecutive spawnings of amphioxus in captivity can be achieved. This has practical implications for the cultivation of other amphioxus species, and eventually will greatly promote the utilization of amphioxus as a model system.

Year-round reproduction and induced spawning of Chinese amphioxus, Branchiostoma belcheri, in laboratory.

Amphioxus is a best candidate for studying the evolutionary and developmental mechanisms of vertebrates, because of its vertebrate-like but much simpler morphology, embryonic development and genome structure. Producing live amphioxus embryos throughout the year is an ideal for comparative evolution and developmental studies. However, all amphioxus species have distinct breeding seasons in the wild and laboratory. We recently found that Chinese amphioxus B . belcheri could reproduce repeatedly beyond its natural breeding season when reared under proper conditions. In this study, we were able to extend further and produce embryos throughout the year from October 2011 to October 2012. We found all examined animals had spawned repeatedly during the examined period. In addition, both lancelets B . belcheri and B . japonicum could be induced to spawn by heat-shock method, although the induced spawning efficiency was not as high as that observed in the European lancelet. In general, we have succeeded in producing B . belcheri embryos almost daily throughout the year. This advancement will provide essential embryonic material for evolutionary and developmental studies, and have great implications for the cultivation and spawning induction of other amphioxus species.

Complete mitochondrial genomes defining two distinct lancelet species in the West Pacific Ocean

Abstract For many years, the lancelet Branchiostoma belcheri had been considered a widely distributed species throughout the West Pacific Ocean. Several recent studies divided it into two separate species based on significant morphological and genetic divergence. However, this taxonomic subdivision has not yet been fully accepted by the scientific community. In order to further clarify the taxonomic status of the West Pacific lancelets, we present here complete mitochondrial genomes (mitogenomes) of two species and compare them at both genomic and phylogenetic levels. These comparisons reveal a significant genetic differentiation and show a deep phylogenetic division. Moreover, to get more insights into the phylogeography of this species complex, we sampled lancelets from Qingdao and Maoming (China) and sequenced their 12S rRNA gene, then combined the data with those from Xiamen and Japan lancelets for phylogenetic analysis. The results were consistent with our previous identification and proved that the lancelets distributed in Qingdao and Japan as well as B. japonicum in Xiamen waters belong to the same species, and those in Maoming and B. belcheri in Xiamen waters are different. Finally, we discuss the implications of these findings on the distribution of lancelets in the West Pacific Ocean.

Evolutionary and functional diversity of green fluorescent proteins in cephalochordates

Green fluorescent protein (GFP) has been widely used as a molecular marker in modern biological research. Before the recent report of one GFP gene in Branchiostoma floridae, GFP family members were cloned only from other two groups of species: Cnidaria and Copepoda. Here we describe the complete GFP gene repertoire of B. floridae which includes 13 functional genes and 2 pseudogenes, representing the largest GFP family found so far. Coupling with nine other GFP sequences from another two species of genus Branchiostoma and the sequences from Cnidaria and Copepoda, we made a deep-level phylogenetic analysis for GFP genes in cephalochordates and found: 1) GFP genes have experienced a divergent evolution in cephalochordates; 2) all amphioxus GFP genes form four main clades on the tree which had diverged before the radiation of the last common ancestor of all extant cephalochordates; 3) GFP genes in amphioxus shared a common ancestor with that in Copepoda rather than being derived from horizontal gene transfer, which indicates that our ancestor was derived from a fluorescent organism and lost this ability after its separation from Cephalochordata, and also makes GFP a rare gene which has a rather unusual evolutionary path. In addition, we also provided evidence indicating that GFP genes have evolved divergent functions by specializing their expression profile, and different fluorescent spectra by changing their emission peaks. These findings spark two interesting issues: what are GFP in vivo functions in cephalochordates and why they are lost in other examined deuterostomes?

Mutagenesis at Specific Genomic Loci of Amphioxus Branchiostoma belcheri Using TALEN Method

Amphioxus, also called lancelet or cephalochordate, is a promising model organism owning to its particularly evolutionary position, simple genome content and comparable body plan to that of vertebrates (Holland et al., 2004; Bertrand and Escriva, 2011). However, use of amphioxus as a model organism has been limited for many years because of lack of an efficient genomic modification method. Recently, several revolutionary gene targeting methods that could induce directed mutations, insertions and deletions at intended target sites, have been developed (Gaj et al., 2013). Among these methods, the transcription activator-like effector nuclease (TALEN) has drawn much interest because of its efficiency in generating target gene alterations, simplicity in vector design and nearly limitless in targeting range (Huang et al., 2011; Miller et al., 2011; Bedell et al., 2012; Lei et al., 2012). Up to date, this method has been shown to be effective in inducing mutations in a broad range of organisms including zebrafish, frog, rat, mouse and human (Tong et al., 2012; Gaj et al., 2013; Liu et al., 2013a), suggesting a great potential use for adopting it in amphioxus genome engineering. Here, we presented the first report of an effective TALEN-mediated genome editing method in Chinese amphioxus, Branchiostoma belcheri. We chose B. belcheri because it is the only amphioxus species which could spawn consecutively all year round (Li et al., 2012, 2013) and be raised through generations in captivity (Zhang et al., 2007). Besides, the species is one of the four amphioxus frequently used in evolutionary/developmental studies and its genome sequence (http://mosas.sysu.edu.cn/genome/gbrowser_wel.php) and embryo microinjection are available in our lab (Liu et al., 2013b).